Musculoskeletal diseases including fractures and osteoporosis are the second-greatest cause of disability worldwide. Unfortunately, our ability to treat or repair damaged bone is extremely limited. This contrasts sharply with human diseases of heterotopic ossification which clearly show that adult humans can form large amounts of bone, particularly after acute trauma. The innate immune system is a key mediator of injury responses and has critical functions in regulating fracture repair and anabolic responses to hormones. Innate immune cells such as macrophages are also important for the formation of heterotopic bone. In this proposal we use a genetic disease of heterotopic ossification, fibrodysplasia ossificans progressiva (FOP), as a model to understand how the immune system affects post-natal bone formation. FOP is characterized by massive heterotopic ossification that can occur after injury. FOP is caused by a mutation in ACVR1 which increases receptor sensitivity to bone morphogenetic proteins (BMPs), but the mechanisms that link injury to ossification are unclear. Our central hypothesis is that activation of the innate immune system can enhance the recruitment and differentiation of skeletal precursors in FOP. We previously created human induced pluripotent stem cells (iPS cells) from patients with FOP. These iPS cells showed increased chondrogenesis and mineral deposition. Our cultures also showed increased numbers of endothelial cells (ECs), which can adopt MSC-like properties when transfected with the FOP ACVR1 mutation. We also found that FOP iPS cell-derived endothelial cell progenitors (iECPs) expressed high levels of OSTERIX, RUNX2, and SOX9, three master regulators of bone formation. We will pursue three aims to determine how the innate immune system is affected by ACVR1 activity in FOP. In Aim 1, we will create a new set of iPS cell lines marked with an OSX/SP7-mCherry reporter to detect osteoprogenitors. We will then test if FOP iECPs exposed to inflammatory cues form more osteogenic precursors than control lines. In Aim 2, we will test if FOP iECP cells are chemotactic to candidate cytokines we identified in our preliminary experiments, and if FOP iPS cell derived macrophages to respond abnormally to activation triggers. Finally, in Aim 3, we will test if macrophage-specific activation of ACVR1 by the Q207D mutation is sufficient to initiate heterotopic ossification after injury in vivo. Togethe, our studies use newly-developed techniques to understand how the BMP signaling pathway activated by ACVR1 leads to heterotopic ossification. The tools and findings are directly applicable to FOP and can also be used to study diseases in and out of the skeleton. Finally, understanding how the immune system can enhance skeletal growth will be useful for treating diseases of bone loss, preventing abnormal bone gain, and improving allograft survival and function.